JEV is a member of the Flaviviridae family and is transmitted by mosquitoes. It is an important human pathogen that causes permanent neuropsychiatric sequelae and even fatal disease, especially in children (Tsai, Vaccine, 2000, 18(Suppl 2), 1-25; Solomon, Neurological Infections and Epidemiology, 1997, 2, 191-199; Umenai et al., Bull. W.H.O., 1985, 63, 625-631). Up to 50,000 cases with a mortality rate of about 25% are reported annually, and about half of the survivors exhibit permanent neuropsychiatric sequelae (Vaughn and Hoke, Epidemiol. Rev., 1992, 14, 197-221; Burke and Leake, Japanese encephalitis, 1988, 63-92, CRC Press Publisher). JEV is distributed mostly in Asia from the former Soviet Union to India. In recent years, however, transmission of the virus has recently been observed in the southern hemisphere, indicating that this virus could become a worldwide public health threat (Hanna, et al., Med. J. Aust., 1999, 170, 533-536; Hanna, et al., Med. J. Aust., 1996, 165, 256-260; Mackenzie et al., Arch. Virol., 1994, 136, 447-467).
JEV is a small-enveloped virus with a single-stranded, positive-sense RNA genome approximately 11 kb in length. The genome contains a single long open reading frame (ORF) flanked by 5′ and 3′ nontranslated regions (NTRs) that are important cis-acting elements for viral replication. The RNA genome of JEV has a type I cap structure at its 5′-terminus but lacks a poly(A) tail at its 3′ terminus. The ORF is translated into a large polyprotein that is co- or posttranslationally processed into three structural and seven nonstructural proteins whose genes are arranged in the genome as follows: C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5 (Lindenbach and Rice, Flaviviridae: The viruses and their replication, 2001, 991-1041, Lippincott Williams&Wilkins Publishers; Venugopal and Gould, Vaccine, 1994, 12, 966-975; Chamber et al., Ann. Rev. Microbiol., 1990, 44, 649-688). Further information, for example, on the function of the majority of the JEV gene products and the molecular mechanisms involved in JEV replication, neurovirulence, and pathogenesis, is limited largely because of the lack of a reliable reverse genetics system.
Research investigating positive-sense RNA viruses has been considerably advanced by the development of the reverse genetics system. Here, infectious cDNA clones of the viral genome in question are constructed and become the templates for infectious RNA synthesis that generates synthetic viruses. There are two approaches, RNA-launched approach and DNA-launched approach, for the reverse genetics system. In the classical “RNA-launched” approach, cells are transfected with RNA transcripts made from the infectious cDNA clones, and the synthetic viruses are then recovered from these cells (Satyanarayana et al., Proc. Natl. Acad. Sci. USA, 1999, 96, 7433-7438; van Dinten et al., Proc. Natl. Acad. Sci. USA, 1997, 94, 991-996; Liljestrom and Garoff, Biotechnology, 1991, 9, 1356-1361; Rice et al., New Biol., 1989, 1, 285-296, Rice et al., J. Virol., 1987, 61, 3809-3819). In an alternative “DNA-launched” approach, synthetic viruses are generated by directly transfecting infectious cDNA clones into susceptible cells. This approach was first reported for poliovirus (Racaniello and Baltimore, Science, 1981, 214, 916-919), and has been adapted for alphaviruses (Schlesinger and Dubensky, Curr. Opin. Biotechnol., 1999, 10, 434-439).
Both of these approaches have been used to construct infectious cDNA clones for many positive-sense RNA virus families, including coronaviruses, which have the largest RNA genomes (Almazan et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5516-5521). These clones have been invaluable in addressing many questions regarding the positive-sense RNA viruses. However, the construction of a full-length infectious cDNA clone for JEV has been hampered, largely because of the genetic instability of the cloned cDNA. Despite extensive efforts, a genetically stable full-length infectious cDNA molecular clone for JEV does not exist (Mishin et al., Virus Res., 2001, 81, 113-123; Zhang et al., J. Virol. Methods, 2001, 96, 171-182; Sumiyoshi et al., J. Infect. Dis., 1995, 171, 1144-1151; Sumiyoshi et al., J. Virol., 1992, 66, 5425-5431).
Thus, the present inventors have disclosed the complete full-length nucleotide sequence of the JEV strain CNU/LP2, isolated from a pool of circulating mosquitoes in Korea. Based on this sequence, the present inventors also have developed a convenient and reliable reverse genetics system for JEV by synthesizing full-length infectious JEV cDNA molecular clones. The reverse genetics system based on the novel infectious JEV cDNA of the present invention can be effectively used for investigating the functions of JEV gene products and other molecular biological mechanisms related to replication, neurovirulence, and pathogenesis of JEV. Further, the present inventors have completed the present invention by confirming that the infectious JEV cDNA can be effectively used as a vector for the heterologous gene expression in a variety of ways.